Method of making a gradient gel with a movable dispensing tip

ABSTRACT

An apparatus and method for making a gradient gel. The present invention in one embodiment is a gel-making system that has a reservoir for holding a solution. The reservoir is connected to a movable arm through a tubing. The tubing has two ends: one end is in fluid communication with the reservoir; and the other, an open end, is received by the movable arm. A gel holder having an internal gel chamber is placed underneath the movable arm for receiving the solution. In operation, the movement of the movable arm causes the open end of the tubing to move along with it and the open end of the tubing delivers the solution in motion to the internal gel chamber to form the gel.

This application is a divisional application of, and claims benefit of,U.S. application Ser. No. 09/219,402, filed Dec. 23, 1998, now U.S. Pat.No. 6,267,579, the disclosure for which is hereby incorporated herein inits entirety by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention comprises an apparatus and method for making agradient gel, and more particularly, the present invention relates to anapparatus and method of using a movable dispensing device to form auniform linear gradient across a wide gel that provides more than twentysample lanes so that more than forty samples can be analyzedsimultaneously with a conventional dual-gel electrophoretic chamber.

2. Background Art

DESCRIPTION OF THE PRIOR ART

Sixty to seventy five percent of the cholesterol in blood is associatedwith low density lipoproteins (“LDL”) which consist of a non-homogeneousmixture of spherical particles ranging widely in particle size (23-28nm), buoyant density and chemical composition. Using a non-denaturing2-16% polyacrylamide gradient gel electrophoresis, researchers havenoted that individuals with a high-risk lipid profile were most likelyto have primarily small, dense LDL particles, as discussed in the paper“Genetic control of low density lipoprotein subclasses,” Austin et al.,Lancet 2: 592-595(3)(1986). In a case-control study of men and womenwith documented myocardial infarction (MI) published in the paper“Low-density lipoprotein subclass patterns and risk of myocardialinfarction,” Austin et al., J. Amer. Med. Assoc. 260: 1917-1921(4)(1988), it was reported that LDL phenotype B, the LDL subclass patterncharacterized by a preponderance of small dense LDL particles, wasassociated with a 3-fold increased risk of MI. This association remainedsignificant after adjustment for age, sex and relative weight. It hasalso been suggested that there may be a major genetic determinant forthis LDL phenotype as in the paper “Inheritance of Low-densitylipoprotein subclass patterns: results of complex segregation analysis,”Austin et al., Am J Hum Genet 73: 838-876 (5) (1988). Whether or not therelationship between LDL phenotype and CAD is independent of other riskfactors such LDLc, HDLc or TRIG is still unclear.

High density lipoproteins (HDL) are responsible for the reversetransport of cholesterol from peripheral tissues back to the liver. Datafrom the paper “Altered particle size distribution of apoA-I-containinglipoproteins in subjects with coronary artery disease,” Cheung et al.,J. Lipid Res. 32: 383-397 (1991), and the paper “Characterization ofhuman high density lipoproteins by gradient gel electrophoresis,”Johansson et al, Biochim Biophys Acta 665: 708-719 (1991), would suggestthat patients with documented CAD may have altered HDL particle sizedistribution when compared to that observed in non-CAD controls. Inthese studies, the heterogeneity of plasma HDL was assessed using anon-denaturing 4-30% polyacrylamide gradient gel first described in thepaper “Characterization of human high density lipoproteins by gradientgel electrophoresis,” Blanche et al., Biochim Biophys Acta 665: 708-719(1981).

A major impediment to large prospective studies of lipoprotein particlesize distribution has been the unavailability of an efficient andreproducible method that can allow the determination of particlediameters for cholesterol-rich lipoproteins. This is mainly because highquality pre-cast gradient gels used in the earlier studies are no longeravailable commercially. The paper “Production of polyacrylamide gradientgels for the electrophoretic resolution of lipoproteins,” Rainwater etal., J. Lipid. Res. 33: 1876-1881 (1992), has reported a procedure forthe preparation of a 4-30% gradient gel which provides estimates of HDLparticle size comparable to those obtained with the PAA 4/30 gel(Pharmacia). In this gradient, however, LDL and larger lipoproteinparticles tend to accumulate at the top of the gel, prohibiting thedetermination of particle size of these lipoproteins. A custom-made2-16% gradient gel was also described by these investigators for thedetermination of LDL particle size in the paper “Effects of diabetes onlipoprotein size,” Singh et al., Arterioscl. Thromb. Vasc. Biol. 15:1805-1811 (1995). Except for Gambert et al., who used lipid staining tovisualize the LDL band as disclosed in the paper “Human low densitylipoprotein fractions separated by gradient gel electrophoresis:Composition, distribution and alterations induced by cholesteryl estertransfer protein,” J. Lipid. Res. 31: 1199-1210 (1990), mostinvestigators used Coomassie to stain the gels for protein after theelectrophoresis. The use of a protein stain typically requires extensivestaining and de-staining procedures for the gels after electrophoresisand special handling of the gels during these steps to maintain gel sizeand shape before scanning. Furthermore, by using a protein stain, manyprotein bands other than those corresponding to plasma lipoproteins arevisible from the electrophoresis of whole plasma.

It is very difficult to make high quality of gradient gels for medicalstudies and clinic use. In the casting of the typical gradient gels, asshown in Rainwater et al. paper, the polyacrylamide solutions arecommonly allowed to flow into a gel chamber from a stationary dispensingtip which is typically placed at the center of the gel. However, as thepolyacrylamide solution flows from the dispensing tip to the sides ofthe plate, a secondary gradient is formed across the width of the gelresulting in lower gel concentrations toward the edges because of thediffusion of the solution. In order to reduce this diffusion effect,only narrow gels with 6-8 lanes across have been available to-datealthough a typical gel chamber is capable of having gels with up to 20or more lanes. Moreover, uneven gradients and disturbances in theprocess of gel making due to the diffusion still exist even in thenarrow gels.

SUMMARY OF THE INVENTION

Definitions

A number abbreviations used in this application for some frequently usedtechnical terms are defined as the following:

The term “S-GGE” as used herein shall refer to a segmental gradient gelelectrophoresis.

The term “S-GGE 2.8/8.30” as used herein shall refer to a 2.8/8.30segmental gradient gel electrophoresis with a 2-8% gradient stackedabove an 8-30% gradient.

The term “LIPOPROTEIN” as used herein shall refer to a class of plasmaproteins that are complexed to lipids.

The term “TRIG” as used herein shall refer to triglycerides.

The term “CHOL” as used herein shall refer to cholesterol.

The term “LDL” as used herein shall refer to low density lipoproteins.

The term “HDL” as used herein shall refer to high density lipoproteins.

The term “Lp(a)” as used herein shall refer to lipoprotein(a) whichconsist of one LDL particle complexed to one apo(a) particle.

The term “LpB” as used herein shall refer to apob-containinglipoproteins.

The term “LDLc” as used herein shall refer to LDL-cholesterol.

The term “HDLc” as used herein shall refer to HDL-cholesterol.

The term “LpA-I” as used herein shall refer to apoA-I containinglipoproteins.

The term “LpA-I/A-II” as used herein shall refer to lipoproteinscontaining both apoA-I and apoA-II.

Summary

The present invention provides a new apparatus and method for making auniform gel including a uniform, continuous gradient gel in many lanesoccupying up to the capacity of a gel chamber. Moreover, the presentinvention can be practiced to produce a segmental gradient gel thatwould provide optimal conditions for the simultaneous characterizationof LDL, Lp(a) and remnant lipoproteins (2-8% gradient) and HDLsubclasses (8-30% gradient) from whole plasma. Additionally, the presentinvention allows the bands corresponding to all of the majorlipid-carrying particles to be visualized without any handling of thegel. The present invention can also be practiced to make severalgradient gels simultaneously. In sum, the present invention offers anew, better, and efficient gel making apparatus and method.

The present invention in one embodiment is a gel-making system that hasa reservoir for holding a solution. The reservoir is connected to amovable arm through a tubing. The tubing has two ends: one end is influid communication with the reservoir; and the other, an open end, isreceived by the movable arm. A gel holder having an internal gel chamberis placed underneath the movable arm for receiving the solution. Inoperation, the movement of the movable arm causes the open end of thetubing to move along with it and the open end of the tubing delivers thesolution in motion to the internal gel chamber to form the gel.

In order to make a gradient gel, normally two solutions with differentconcentrations are used. Accordingly, one embodiment of the presentinvention employs a gradient maker that consists of a reservoir having afirst container and a second container. The first container holds afirst solution and the second container holds a second solution. Achannel connects the first container and the second container with anoutlet connected to the second container and in communication with thechannel so that a fluid of the first solution and the second solution isformed at the outlet. A tubing having a first end and a second endconnects to the outlet with the first end. The second end of the tubingis received by a movable arm and moves along with the movable arm. A gelholder with an internal gel chamber is placed underneath the movable armfor receiving the fluid, where the chamber has a longitudinal axis. Themovable arm moves back and forth along the longitudinal axis so that thesecond end of the tubing delivers the fluid in motion in the gel chamberto form the gradient gel. In a linear gradient gel the bottom of the gelhas a higher concentration and the top of the gel has a lowerconcentration.

A linear gradient gel can be formed from more than two solutions. Inanother embodiment of the present invention, a reservoir has a pluralityof containers holding a plurality of solutions. Each container holds onesolution and communicates with at least one neighboring container. Anoutlet is connected to at least one container to communicate with thecontainers so that a fluid of at least two solutions from the pluralityof solutions is formed at the outlet. A tubing, having a first end and asecond end, is connected to (and is in fluid communication with) theoutlet through the first end. A movable arm receives the second end ofthe tubing and causes the second end of the tubing to move along withit. A gel holder with an internal gel chamber is placed underneath themovable arm for receiving the fluid, where the chamber has alongitudinal axis. The movable arm moves back and forth along thelongitudinal axis of the chamber so that the fluid is transferred fromthe first end to the second end of the tubing and then is delivered inthe chamber by the second end of the tubing in motion to form thegradient gel.

In gel making, approximately two hours are required for the gel solutionto polymerize and form a solid matrix. One advantage of the presentinvention is that several highly uniform gradient gels can be madesimultaneously. In one embodiment of the present invention, a pluralityof reservoirs are utilized. Each of them has a first container and asecond container, where the first container holds a first solution andthe second container holds a second solution. A channel connects thefirst container and the second container. Moreover, an outlet isconnected to the second container and communicates with the channel.Consequently, a fluid of the first solution and the second solution isformed at the outlet of this particular reservoir. Thus, a plurality offluids are formed at the plurality of the outlets of the plurality ofthe reservoirs. Furthermore, a plurality of tubings, each having a firstend and a second end, connect in a one to one relationship to theplurality of reservoirs through the connection of the second end of atubing to the outlet of a reservoir. A movable arm carries at least aplurality of the second ends of the plurality of the tubings. And aplurality of gel holders are positioned in parallel thereby defining alongitudinal axis. Each of the gel holders has an internal gel chamberfor receiving the fluid from one of the plurality of the second ends.When the movable arm moves back and forth along the longitudinal axis,each second end of the tubings delivers one of the fluids in motion intoone gel chamber to form one gradient gel. As a result, a plurality ofthe gradient gels are produced.

In order to make an uniform gradient gel, the movable arm moves at asubstantially constant rate of motion. While other mechanisms may beused, one embodiment of the present invention uses a motor to drive themovable arm. One advantage of using the motor driving mechanism is thatby adjusting the speed of the motor, the rate of motion of the movablearm can be selected. In order to ensure that the same gel concentrationis present across the width of the gel, the rate of motion of themoveable arm is adjusted and set according to the width of the gel andthe rate of flow of the solution from the reservoir into the gelchamber. For instance, for a wider gel, the rate of motion should beincreased to cover a greater distance in the same interval of time.Also, the height of the reservoir relative to the gel chamber can affectthe flow rate, which can be readily taken into account when setting therate of motion of the movable arm. According to the preferredembodiment, the movable arm has an internally threaded bore and themotor controls the motion of the movable arm through a shaft. The shafthas an elongated body with an external thread on the elongated body. Theshaft has a longitudinal axis and is rotatable around its longitudinalaxis. The shaft can rotate around the longitudinal axis either inclockwise direction or counter-clockwise direction, and the direction ofrotation of the shaft is changeable from clockwise to counter clockwise,or vice versa. The external thread on the elongated body is adapted tomate with the movable arm through the internally threaded bore. Theshaft operatively connects to the motor. Thus, when the motor causes theshaft to rotate around its longitudinal axis, the movable arm movesalong the longitudinal axis because the mating mechanism of the externalthread of the shaft with the internally threaded bore of the movablearm. Because the shaft can rotate around the longitudinal axis either inclockwise direction or counter-clockwise direction, the movable arm isable to move along the longitudinal axis both forward and backward. Aswitching mechanism may be used to control the direction of the rotationof the shaft.

The movable arm in turn receives an open end of the tubing, whichconnects with the reservoir at the other end, the open end moving withthe movable arm. To do so, the movable arm has means for holding atleast a portion of the tubing proximate to the open end of the tubing sothat at least the open end of the tubing moves along with the movablearm. In one embodiment of the present invention, the holding means is anopening sized to allow the open end of the tubing to pass through buthold at least the portion of the tubing proximate to the open end of thetubing therein. The movable arm may have several openings at differentlocations to allow several gels to be made simultaneously, or to give auser freedom to set up the user's devices. The openings can also havedifferent sizes to accommodate tubings with different sizes.Alternatively, other holding means, including a clamping device normallyused in laboratories such as a clamp, can be used to associate thetubing with the moveable arm.

The tubing is made from a flexible material so that it can move alongwith the movable arm easily without impeding the flow of the fluidwithin the tubing. Many materials can be used. In one embodiment of thepresent invention, tubings made from Manosie silicone rubber by VWRScientific Products Corporation, located at Willard, Ohio are used. Inuse, the tubing transfers fluid from the reservoir and delivers thefluid in motion at a substantially constant rate of flow. The tubing maydeliver the fluid through a dispensing tip. Or, the fluid can bedelivered simply through an open end of the tubing.

Solutions to make a linear gradient gel normally are polyacrylamidesolutions. These solutions can be identified as high concentrationpolyacrylamide solution, medium concentration polyacrylamide solutionand low concentration polyacrylamide solution. For a preferredembodiment of the present invention, a solution according to a mixingratio of a 2.3 g acrylamide and 0.1 g N,N′-methylene-bis-acrylamide in100 ml of borate buffer is regarded as a low concentrationpolyacrylarnide solution (2.4%), a solution according to a mixing ratioof a 10.24 g acrylamide and 0.43 g N,N′-methylenebis-acrylamide in 100ml of borate buffer is regarded as a medium concentration polyacrylamidesolution (10.67%), and a solution according to a mixing ratio of a 38.4g of acrylamide and 1.6 g N,N′-methylene-bis-acrylamide in 100 ml ofborate buffer is regarded as a high concentration polyacrylamidesolution (40%).

These solutions have been used successfully in the present invention toproduce high quality segmental linear gradient gels, namely, a 2-8%continuous linear gradient gel and a 8-30% continuous linear gradientgel. In doing so in one embodiment of the present invention, acommercial gradient maker with a container A and a container B, such asa Hoefer GS 100, purchased from Hoefer Scientific Instr., San Francisco,Calif. is loaded with solutions. Container A and container B connect toeach other through a channel. An outlet connects to the container B andcommunicates with the channel so that a mixed fluid of the firstsolution and the second solution is formed at the outlet. For thisembodiment, the higher concentration of solution is always in containerB. To make a 8-30% linear gradient gel, a high concentration ofpolyacrylamide is in container B and a medium concentration ofpolyacrylamide is in container A. A tubing transfers the mixed fluid ofthe high concentration of polyacrylamide and the medium concentration ofpolyacrylamide from the outlet to a dispensing tip. The dispensing tipcan be a separate device. Or an open end of the tubing can function asthe dispensing tip. A movable arm receives the dispensing tip. A gelholder having an internal gel chamber with a longitudinal axis is placedunderneath the dispensing tip. The motion of the movable arm along thelongitudinal axis causes the dispensing tip to move along with movablearm and the dispensing tip delivers the fluid in motion in the gelchamber. Because the motion of the movable arm can be controlled at asubstantially constant rate, the fluid can be evenly deliveredthroughout the gel chamber. Moreover, because the movable arm is capableof traveling the length of the gel chamber, the solution is delivereddirectly to the gel chamber “on-the-spot.” Thus, a secondary gradientresulting from the diffusion of the solution from the dispensing tip tothe edge of the gel is not formed. After a proper curing period, auniform, high quality 8-30% gradient gel is produced. Since in thesegmental linear gradient gel a second gel (2-8% segment) must be pouredon top of the first gel (8-30% segment), the volume of the solutions iscontrolled so that the 8-30% gradient gel occupies half space of the gelchamber. As people skilled in the art appreciate, the exact volumes tobe placed in the reservoir can be calculated from the dimension of thegel chamber before hand. Furthermore, the entire gel making processaccording to the present invention can be automated by placing the rightvolumes of the solutions in the reservoir. The gel is poured tocompletion until the reservoir is emptied.

The containers A and B are subsequently filled with a mediumconcentration of polyacrylamide (in container B) and a low concentrationof polyacrylamide (in container A) to make a 2-8% gradient gel. Theabove process is then repeated. And a uniform 2-8% gradient gel isformed on top of the 8-30% gradient gel. As a result, a segmental lineargel consisting of two continuous linear gradients is formed, which canthen be used for the simultaneous determination of the diameters of LDLand HDL from whole plasma. The matching of the concentrations at theinterface of the two linear gradients in this embodiment provides acontinuous transition between the two linear gradients.

In a further embodiment of the present invention, two or more gradientmakers, each with two containers A and B, can be used to simultaneouslymake two or more gradient gels in two or more gel chambers. Therefore,practicing the present invention is economic and efficient, in additionto the advantages of making better continuous gradient gels.

In an additional further embodiment of the present invention, multiplelinear gradients can be stacked on top of another to allow optimalseparation of the macromolecules of interest. For example, the presentinvention can be practiced to make a linear gradient gel with threesegments. Similarly, gels having other concentration gradients can alsobe made.

Other objects, advantages and uses for the present invention will bemore clearly understood by reference to the remainder of this document.

BRIEF DESCRIPTION OF THE FIGURES OF THE DRAWINGS

FIG. 1 is a schematic view of a gel making system according to thepresent invention.

FIG. 2 is a schematic view of a first alternative embodiment of the gelmaking system of the present invention.

FIG. 3 is a schematic view of a second alternative embodiment of the gelmaking system of the present invention.

FIG. 4 is a partial side view of a gel making system of the presentinvention.

FIG. 5 is a partial top view of the gel making system shown in FIG. 4.

FIG. 6 is a perspective view of a movable arm according to a preferredform of the present invention.

FIG. 7 is a cross-sectional side view of the movable arm shown in FIG.6.

FIG. 8 shows reproducibility of lipoprotein particle sizedeterminations: (A) an actual photograph of a gradient gel with the sameplasma sample (CHOL=279, TG=65, HDLc=65 and Lp(a)=175 mg/dL) beingapplied to 17 of the 21 available lanes. Aliquots of this plasma samplewere also applied to different lanes (x-axis) for 5 different gels(different symbols) over a two-week period; (B) the calculated particlediameters (nm) are plotted on the vertical axis as function of lanenumber (horizontal axis) for each gel and the different symbolsrepresent results from different gels. The mean diameter (-), 1 SD range(---) and 2 SD range (--) are presented for LDL and Lp(a) particlediameter; and (C) same as (B) but for HDL₂ and HDL₃.

FIG. 9 identifies conditions for optimal lipoprotein bands on the S-GGE2.8/8.30 gel (‘S’ indicates the lane to which the Standard (calibrator)sample was applied): (A) by varying the incubation period forpre-staining of whole plasma from 120 min (Lane 1), 60 min (Lane 2), 30min (Lane 3) to 15 min (Lane 4) it is showed that there was no effect onthe position of the Lp(a), LDL, HDL₂ and HDL₃ bands; (B) the position ofthe LDL band was not affected by the concentration of LDLc in the 10 μlof sample applied to the lane. A concentrated preparation of LDL(LDLc=270 mg/dL, Lane 5) isolated by ultracentrifugation was dialyzedagainst normal saline (d: 1.006 and 0.01% EDTA) and diluted with d:1.006 density solution containing 0.01% EDTA to various concentrationsof cholesterol ranging from 160 (Lane 1), 80 (Lane 2), 50 (Lane 3), to40 (Lane 4) mg/dL. All diluted samples were pre-stained by incubation atroom temperature for 2 hours; and (C) the application of comparableconcentrations of cholesterol in the form of VLDL resulted inconsiderably broader bands which are less intensely stained as comparedto LDL and Lp(a). VLDL was isolated by ultracentrifugation and appliedat cholesterol concentrations of 100 mg/dL (Lane 1, ˜530 mg/dL of TG),50 mg/dL (Lane 2, 265 mg/dL of TG) and 25 mg/dL (Lane 3, 133 mg/dL ofTG).

FIG. 10 shows gel scans for whole plasma, isolated Lp(a) and isolatedLDL, where Plasma from a healthy normal control woman (CHOL=279, TG=65,HDLc=65, and Lp(a)=175 mg/dL) was used for this experiment: (A) twosharp and distinct peaks can be visualized corresponding to lipoproteinparticles with diameters of 23 nm or greater. In view of the low plasmaTG and high Lp(a) levels in this individual, we postulated that thispeak of larger diameter corresponded to Lp(a); (B) By density gradientultracentrifugation, a fraction enriched in Lp(a) was isolated andconfirmed by ELISA. Upon electrophoresis, this fraction exhibited amajor band at the same position as the ‘designated Lp(a)’ when wholeplasma was used. There was a minor peak of small LDL which can bevisualized. This would be consistent with contamination by LDL of theLp(a) fraction which was isolated at a density higher than plasma LDL;and (C) LDL isolated in the density range d: 1.020-1.063 from the sameplasma exhibited a single peak corresponding to the major LDL peak seenwith whole plasma.

FIG. 11 shows gel scans of whole plasma samples obtained at differenttimes following the consumption of a fat-containing meal: (A) analysisof fasting plasma from a female patient with documented CAD (Lp(a)=85,CHOL=175, TG=125, HDLc=79 mg/dL). In addition to the peaks correspondingto Lp(a) (Peak 2), LDL (Peak 3), HDL₂ (Peak 4), HDL₃ (Peak 5) andalbumin (Peak 6), this sample had a prominent shoulder (Peak 1) to theleft of the Lp(a) peak suggestive of the presence of largerlipoproteins; (B) same as (A) but after 2 hours showing a well-definedpeak; (C) same as (A) but after 4 hours; (D) same as (A) but after 10hours, showing Peak 1 became more of a shoulder to the left of the Lp(a)peak as was the case with the fasting plasma sample. This broad peak isbelieved larger than Lp(a) represents TG-rich remnants. The diameter ofPeak 1 is estimated at 36.7 nm with a range of 32 to 39 nm in differentsamples.

FIG. 12 shows comparison of HDL particle size with the SFBR 3/31 gelsystem: (A) displays a photograph of a gradient gel depicting thecalibrator (Lane 1), whole plasma (Lane 2), LpB (Lane 3), LpA-I (Lane 7)and LpA-I/A-II (Lane 5) for one subject. The band in Lane 7 identifiedin the size range of plasma LDL does not contain apoB as determined byELISA and can be shown by FPLC to contain phospholipids, cholesterylesters, cholesterol, apoE and apoC's (data not shown); (B) showscorrelation between HDL diameters determined by protein staining usingthe SBFR 3/31 gel from Alamo Gels Inc. and by lipid staining using theS-GGE 2.8/8.30 gel after the diameters of the HDL calibrators wereadjusted to be comparable with the values reported by Northwest LipidResearch Laboratory. The triangles (D) correspond to peaks identifiedfrom LpA-I fractions and the circles (O) indicate peaks identified fromLpA-I/LpA-II fractions.

FIG. 13 shows the effect of sample storage on LDL particle diameter,where plasma samples from 51 subjects including normolipideric controlsand patients with various forms of dyslipidemia were electrophoresedfresh (within 7 hrs of sample collection) and after storage at −80° C.(3 to 12 months in cryovials without any additives): no statisticallysignificant difference was found between the two estimates by two-tailedpaired t-test. The ratio of the two estimates of LDL particle diameters(Fresh/Frozen) is plotted as a function of LDL particle diameterdetermined from freshly isolated plasma. The mean ratio (-), 1 SD (---)and 2 SD lines (--) are also plotted. The variability in the ratioreflects the combined effect of sample storage and gel reproducibility.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is more particularly described in the followingexamples that are intended as illustrative only since numerousmodifications and variations therein will be apparent to those skilledin the art. As used in the specification and in the claims, “a” can meanone or more, depending upon the context in which it is used. Thepreferred embodiment is now described with reference to the FIGS. 1-13,in which like numbers indicate like parts throughout the FIGS. 1-13.

OVERVIEW

Referring generally to FIGS. 1-7, the present invention comprises a gelmaking system that utilizes a movable dispensing mechanism and method tomake high quality gels, especially continuous gradient gels. The presentinvention allows the reproducible preparation of gradient gels which canbe as wide as desired in order to accommodate any number of samplessimultaneously. One embodiment of the present invention accommodates upto 21 samples per gel, or 42 samples per run with a dual-gelelectrophoretic chamber, compared to several samples per gel usingcurrently available gel making devices. The present invention allows theapplication of pre-stained plasma samples and the gel can be scanned forparticle size immediately at the end of the electrophoretic procedure.Elimination of the extensive staining and de-staining steps afterelectrophoresis should also minimize the need to handle the gelpreventing any artifact that these steps may introduce. Equallyimportant, the use of a lipid stain allows the specific visualization ofonly the lipoprotein fractions present in whole plasma. Thus, thepresent invention provides a new device and method to facilitate medicalresearch in the related fields.

Referring now to FIG. 1, the gel making system 100 of the presentinvention, according to one preferred embodiment, has a reservoir 10 forholding gel making solutions, a tubing 20, a movable arm 30, and a gelholder 50. The reservoir 10 has two containers 12, 14, each of them isused for holding one solution. As shown in FIG. 1, container 12 holdssolution A and container 14 holds solution B. The containers 12 and 14are connected by a channel 16 so that the containers can be in fluidcommunication to each other. Cross-sectionally, channel 16 can besquare, oval, circular or in other geometrical shapes. An optionalstopcock or valve 18 may be used to control the fluid communicationbetween the containers 12, 14. Moreover, as people skilled in the artknow, the ratio of the cross-sectional dimension of the channel 16 tothe cross-sectional dimension of an outgoing channel 15 determines thecharacteristic of the gradients: a linear gel is generated when they arethe same and a nonlinear gel results when they are not.

An outlet 19 is connected to the outgoing channel 15 and communicateswith the channel 16 so that a mixture of the solution A and the solutionB is formed by the time the solutions reach the outlet. Thecross-sectional area of the channel 16 determines how fast solution A incontainer 12 mixes with solution B in container 14. The location of theoutlet 19 is important but variable. In the embodiment shown in FIG. 1,the outlet 19 is connected to the container 14. Alternatively, theoutlet 19 can be connected to the container 12. Or the outlet 19 may beconnected to both of the containers 12, 14, for example, through thechannel 16, at a location near the stopcock 18. The location of theoutlet 19 impacts the arrangement of solutions in the containers 12, 14.For the embodiment shown in FIG. 1, container 14 always holds a solutionwith a higher concentration of polyacrylamide.

Reservoir 10 can have alternative arrangements of containers. FIG. 2shows one alternative to the embodiment of the reservoir 10 shown inFIG. 1. In FIG. 2, reservoir 10′ has three containers 12, 13 and 14,each of them for holding one solution. As shown in FIG. 2, container 12holds solution A, container 13 holds solution B and container 14 holdssolution C. The containers 12, 13 and 14 are connected by a channel 16and an outgoing channel 15 so that the containers can be in fluidcommunication to each other. Optional stopcocks or valves 17 may be usedto control the fluid communication among the containers 12, 13 and 14 sothat a variety of fluids, such as A+B, A+C, B+C, or A+B+C, can be made.

Multiple reservoirs 10 can also be used in the present invention asshown in FIG. 3, where two reservoirs 10 are used to provide capacityfor making two gradient gels simultaneously as discussed in detail videinfra.

Still referring to FIG. 1, the tubing 20 has two ends 21, 23. The tubing20 is connected to the outlet 19 through the end 21 and is in fluidcommunication with the outlet 19. End 23 is an open end and is receivedby the movable arm 30.

The movable arm 30, referring to FIGS. 1, 6 and 7, has a body 32 andopenings 36 a, 36 b. Each opening is sized to allow the end 23 of thetubing to pass through and hold at least a portion of the tubing 20proximate the end 23 of the tubing therein. In the embodiment shown inFIG. 1, the tubing 23 is received by opening 36 a. Alternatively, it canbe received by opening 36 b. If multiple reservoirs are used as shown inFIG. 3, each opening would receive a tubing. The movable arm 30 can haveone, or two, or more openings to accommodate the need of a user.Moreover, each opening can be sized differently to accommodate the sizeof the tubing. Furthermore, cross-sectionally each opening may becircular, square, rectangular, triangular, diamond, oval or othergeometrical shape. Accordingly, openings can be cylinders (as shown inFIG. 7), cubic, cone or other geometric shapes. The movable arm 30 canalso take different shape. Cross-sectionally the body 32 may becircular, square, rectangular, triangular, diamond, oval or othergeometrical shape. Other alternatives can also be used to associate thetubing 20 with the movable arm 30. For example, a clamp (not shown) canbe used to associate the tubing 20 with the movable arm 30.

A gel holder 50 is placed underneath the end 23 and the movable arm 30.The gel holder 50 has an internal gel chamber 51 defined by two spacers52, 54, two glass plates 56, 58, and a bottom 60. The chamber 51 has anopen top 53 to allow the fluid to be delivered therein through the end23 of the tubing 20. The fluid can be delivered into the chamber 51directly through the open end 23 of the tubing 20. Optionally, adispensing tip (not shown) can be used to deliver the fluid.

The movable arm 30, in this embodiment, is driven by a motor 40. Oneadvantage of using the motor 40 to drive the movable arm is that byadjusting the speed of the motor 40, the rate of motion of the movablearm can be selected. Once a rate of motion is decided, the motor 40 cankeep the movable arm 30 moving at that rate of motion constantly. A widerange of motors may be used. For the preferred embodiment of the presentinvention, motor 40 is an IG P/N 13556-165730 motor manufactured byIgurashi Electric Works, Japan.

The motor 40 controls the motion of the movable arm 30 through a shaft70.

According to the embodiment shown in FIGS. 4-6, the movable arm 30 hasan internally threaded bore 34. The shaft 70 has an elongated body 72with an external thread 74. The external thread 74 on the elongated body72 is adapted to mate with the movable arm 30 through the internallythreaded bore 34. The shaft 70 has a longitudinal axis L and isrotatable around its longitudinal axis. The shaft 70 can rotate bothclockwise and counterclockwise. The direction of the rotation of theshaft 70 is controlled by switches 92 a, 92 b. Switches 92 a, 92 bchange the direction of rotation of the shaft 70 from clockwise tocounter-clockwise, or vice versa. Switches 92 a, 92 b may be pressuresensors, optical-electrical device, electromagnetic device or otherdevices. In this embodiment, switches 92 a, 92 b are double pole, doublethrow type 8221 SHZGE switches, which can be found in most hardwarestores. The shaft 70 operatively connects to the motor 40. Thus, whenthe motor 40 causes the shaft 70 to rotate around its longitudinal axisL, the movable arm 30 is driven to move along the longitudinal axis Lbecause the mating mechanism of the external thread 74 of the shaft 70with the internal thread 35 of the internally threaded bore 34 of themovable arm 30. Because the shaft 70 rotates around the longitudinalaxis either in clockwise direction or counter-clockwise directionalternately, the movable arm 30 is able to move along the longitudinalaxis forward and backward continuously. Other mechanisms can be used tocontrol the motion of the movable arm 30. For example, an air gauge canbe used to drive the movable arm 30. Additionally, electromagneticmechanism and spring mechanism may also be utilized.

The movable arm 30, the motor 40 and the shaft 70 are supported by ahousing 80. Referring now to FIGS. 4 and 5, housing 80 has a horizontalbase 82 with corners 84 a-d. Two supporting posts 86 a, 86 b are bearingagainst the comers 84 a, 84 b. Crossing then horizontal base 82, asupporting board 87 is bearing against the comers 84 c, 84 d.Alternatively, supporting board may be replaced by more supportingposts. Moreover, the positions of the supporting board 87 and thesupporting posts 86 a, 86 b are interchangeable. The supporting posts 86a, 86 b are connected by a horizontal bar 88 a, and the supporting board87 is connected to the supporting posts 86 a, 86 b by horizontal bars 88b, 88 c. In the embodiment shown in FIGS. 4 and 5, the horizontal bars88 b and 88 c are parallel to each other and therefore define alongitudinal axis. The supporting board 87 and the horizontal bar 88 aare parallel to each other but perpendicular to the longitudinal axis.Optionally, the horizontal bars 88 a-c are movably connected to thesupporting posts 86 a, 86 b and the supporting board 87 so that theheights of the horizontal bars 88 a-c relative to the horizontal baseare adjustable individually. The shaft 70 connects the horizontal bar 88a and the supporting board 87 and divides the housing 80 into twocompartments 90 a, 90 b. The motor 40 is operatively connected to theshaft 70 and also supported by the horizontal bar 88 a. The switches 92a, 92 b are mounted on the horizontal bar 88 a and the supporting board87 respectively.

Each of the compartments 90 a, 90 b can be used to receive a gel holder50. Or two gel holders 50 can be received at once. Alternatively, byexpanding the size of the housing 80 and length of the movable arm 30,more gel holders can be placed within the housing 50 to make gels.Moreover, the height of the movable arm 30 relative to the horizontalbase 82 may be adjusted by adjusting the heights of the horizontal bars88 a-c to accommodate gel holders 50 with different heights.

Now the gel making system 100 is ready to be used. Referring now toFIGS. 1, 4 and 5, the solution A from the container 12 and the solutionB from the container 14 form a mixture fluid at the outlet 19. The fluidtravels through the tubing 20 to the open end 23. The motor 40 isactivated to drive the shaft 70 to rotate about the longitudinal axis L.The shaft 70 in turn, by the mating mechanism between the shaft 70 andthe movable arm 30, causes the movable arm 30 to move along thelongitudinal axis. When the movable arm reaches either switch 92 a orswitch 92 b, the rotational direction of the shaft 70 is changed fromclockwise to counter-clockwise, or vise versa. Because the change of thedirection of rotation of the shaft 70, the movable arm reverses it'sdirection of motion along the longitudinal axis as well. As a result,the movable arm moves back and forth between the horizontal bar 88 a andthe supporting board 87. In a continuous motion, the movable arm travelsback and forth along the longitudinal axis and causes the open end 23 ofthe tubing 20 to move along with it. During this oscillating motion, theopen end 23 delivers the fluid into the internal gel chamber 51 throughthe open top 53 between the two spacers 52 and 54. Because the movablearm 30 moves at a constant rate of motion, open end 23 delivers thefluid in motion at a substantially constant rate of flow. After a properamount of the fluid is deposited in the gel chamber 51, it can be curedto form the desired continuous gradient gel.

It is possible to make a second gradient gel on top of the newly madegradient gel so that the two gels form a continuous concentration changefrom the bottom of the first gradient gel to the top of the secondgradient gel. To do so, the volumes of the solutions A and B areselected so that the gradient gel is formed at the bottom half of thegel chamber 51. Then the containers 12, 14 are emptied and refilled withtwo new solutions and the above process is repeated to form a second gelon the top half of the gel chamber 51. Because the fluids are deliveredat substantially constant rate of motion and from side-to-side, theupper edge of the first gradient gel is rather smooth so as to supportthe second gel on the top half. Because the diffusion effect isminimized in the present invention, much wider and better quality gelsare produced according to the present invention.

While it is desirable to make a gel up to the capacity of the gelchamber by delivering the fluid in side-to-side motions, an optionalstopper can be put on the track of the movable arm 30 to customize thesize of the gel to be made. Furthermore, referring to FIGS. 3-5, two ormore gel holders can be placed in the housing 80 to simultaneously makemultiple gels. The solutions at each reservoir can be same or different,depending on a user's need.

The invention will be better understood by reference to the followingillustrative example, which is performed according to the presentinvention.

EXAMPLES

Materials

Polyacrylamide was obtained from Sigma Chemicals (A-3553 and M-7279), aswere the ammonium persulfate (A-1433) and TEMED (T-9281) and the stain,Sudan Black B (Sigma: S-0395). Ethylene glycol (E178-1) and boric acid(BP 168-500) were purchased from Fisher Scientifics.Tris(hydroxymethyl)aminomethane (EK-1174952) was from VWR Scientifics.

Equipment

Some components of the gel apparatus as shown in FIGS. 1-7 werepurchased from Hoefer Scientific Instruments at San Francisco, Calif.and included the SE 650 vertical slab unit, the SG-100 gradient maker,and the PS-1500 power supply. The gradient gel was prepared using the18×8 slab gel unit from Hoefer (SE 6402) with a 3-mm spacer. Two 12-slotsample applicators from Isolabs (GC-50) were adapted for our gel systemto optimize sample loading. The gel was scanned using the LKB 222-020UltroScan XL Laser Densitometer. The oscillating motion of thedispensing arm was controlled by a platform mixer (Vari-Mix,Thermolyne). The movable arm and mating shaft were manufactured in theinventors' laboratory.

Buffers

Three stock preparations containing high, intermediate and lowconcentrations of polyacrylamide were available: (1) HIGH: 38.4 grams ofacrylamide and 1.6 g N,N′-methylene-bis-acrylamide in 100 ml of boratebuffer (80 mM boric acid, 90 mM Tris, 3 mM EDTA, pH 8.3) and (2) MEDIUM:10.24 g acrylaride and 0.43 g N,N′-methylenebis-acrylamide in 100 ml ofborate buffer and (3) LOW: 2.3 g acrylamide and 0.19 gN,N′-methylene-bis-acrylamide in 100 ml of borate buffer. A stocksolution of TEMED was prepared by combining 0.6 ml of TEMED and 99.4 mlof the borate buffer. Ammonium persulfate solution (5 mg/ml) was alsoprepared with the borate buffer. Preparation of Segmental GradientPolyacralamide Gels (S-GGE 2.8/8.30) The segmental polyacrylamidegradient gel (3 mm thick) was poured in two steps using the Hoefer SE6402 gel maker kit with 18×8 cm glass plates and the gradient maker(Hoefer GS 100). Each container of the gradient maker was filled with amixture of 6 ml of the appropriate polyacrylamide concentration, 1 ml ofTEMED buffer and 1 ml of ammonium persulfate solution. For the firststage, the two containers held contained the high (Container B) andmedium (Container A) concentrations of polyacrylamide to generate the8-30% gradient. As shown in FIG. 1, the polyacrylamide solutions wereallowed to flow continuously as the movable arm was moved from side toside along the upper rim of the glass plates, thus creating multipletracks along the glass plates across the width of the gel chamber. Thismotion (15 side-to-side oscillations/min) ensured the even distributionof the polyacrylamide solution between the plates over the entire widthof the gel (14 cm). The containers were allowed to empty completelycreating the lower gradient gel (approximately 3.8-4 cm in height) whichwas then allowed to polymerize at room temperature (2 hours). Thecontainers of the gradient makers were subsequently filled with themedium (container B, 6 ml) and low (container A, 6 ml) concentrationpolyacrylamide to form the 2-8% linear gradient gel. To each container,1 ml of TEMED buffer and 1 ml of ammonium persulfate solution were addedfor a total of 16 ml for both chambers. The quality and reproducibilityof the gel is optimal if the rate of gel flow is matched to theappropriate rate of movement of the dispensing arm. The rate of flow iscontrolled by the mating mechanism between the shaft 70 and the movablearm 30 movable with a mean rate of 1.5 ml/min and the moving arm movedat a rate of 15 cycles/min.

Electrophoresis

Whole plasma (30 μl) was mixed with 10 μl of a prestaining solution(0.6% Sudan Black B solution in ethylene glycol) prior to application tothe gel. After a 2-hour incubation period at room temperature, a singleload of 10 μl of prestained plasma solution was applied to individualtroughs, characteristic of the GA-50 sample applicator (Isolabs).Electrophoresis was performed at room temperature, 50 mA, 80 V for 18-20hours. Alternatively, the gel image can be digitized using the ImageMaster Video Documentation System and Analysis Software, which iscommercially available.

Calibration of Lipoprotein Particle Size

Whole plasma from a human donor exhibiting distinct lipoprotein bandscorresponding to LDL, Lp(a), HDL₂ and HDL₃ (CHOL=297, TG=97, HDLc=73 andLp(a)=29 mg/dL) was run in multiple lanes of each gel. The diameter ofthe LDL band in this plasma sample was calibrated using a set ofin-house LDL calibrators (27.7, 25.3 and 24.2 nm). The diameters for thein-house LDL calibrators had been previously standardized against thecalibrator pool ILH containing LDL with diameter of 29.7, 27.1 and 24.7nm available from Dr. Krauss (Donner Laboratory) as previously reported(16,17) using PAA 2/16 gels from Isolabs.

As with other gradient gel systems, the diameters of HDL₂ and HDL₃ inthe SGGE 2.8/8.30 gradient gel were determined using the high molecularweight calibrators obtained from Pharmacia (HMW 17-0445-01). For thesecalibration runs, the gels were stained for protein with Coomassie afterelectrophoresis to visualize the protein bands corresponding to themolecular weight standards. The high molecular weight calibratorsincluded: thyroglobulin (17.0 nm), ferritin (12.2 nm), catalase (10.4nm), lactate dehydrogenase (8.4 nm), and albumin (7.1 nm). Frozenaliquots of plasma from this donor were maintained at −80° C. (up to 2years) and included in all subsequent runs as calibrators for the gel.The quality of the calibrators was assessed by the actual position ofthe bands as well as the values calculated for the gel constant. Thesealiquots were thawed once for each use and discarded without beingre-frozen for later use.

Determination of Particle Size using the Gel Constant

From gel chromatography experiments, it is known that the pore size(S_(pore)) of polyacrylamide gel varied inversely to the monomerconcentration (T_(gradient)) i.e.

S _(pore) =K ₁[1/T _(gradient)]  [Eq. 1]

where K₁ denotes the unknown proportionality constant. For a lineargradient, the monomer concentration T_(gradient) of the polyacrylamideat any distance d in the gel is a function of the distance d from thetop of the gel, thus

T_(gradient) =f(d).  [Eq. 2]

The function f will also depend on the range and slope of the gradient.By combining Equations 1 and 2, we obtained:

S _(pore) =K ₁×[1/f(d)]  [Eq. 3]

When gradient electrophoresis is carried out to completion, sphericalparticles of uniform size will continue to migrate until they reach adistance in the gradient, d_(particle), where the pore size of the gelmatrix becomes so small as to prevent further penetration by theparticle. At this point of equilibrium the pore size is approximatelyequal to the particle size. In other words,

S _(particle) =S _(pore) =K ₁×[1/f(d _(particle))]  [Eq. 4]

or

C _(gel) =S _(particle) ×f(d _(particle))=S _(pore) ×f(d_(particle))  [Eq. 5]

By using a calibrator of known particle diameter S_(calibrator) and bymeasuring the distance d_(calibrator) migrated into the gel of thiscalibrator, we can calculate the gel constant, C_(gel), for thisparticular linear gradient.

C _(gel) =S _(calibrator) ×f(d _(calibrator)).

Using the distances determined by the gel scanner for the bandscorresponding to the LDL, Lp(a), HDL₂ and HDL₃ calibrators, we cancalculate the gel constants for the upper and lower gels. Since thedistance d_(calibrator) depends ultimately on the polyacrylamideconcentration in the gel gradient, Eq. 5 would indicate that C_(gel)will depend only on the pore size and should, in theory, be the sameindependent of the gradient:

C _(gel) (nm-%)=S _(calibrator) ×[L+(d_(calibrator))*(Slope_(gradient))]  [Eq. 6]

where S_(calibrator) is the known diameter (nm) of the calibrator

L is the lowest gel concentration for the gradient, i.e. 2% for the 2-8%gradient and 8% for the 8-30% gradient

d_(calibrator) (mm) is the position of the band corresponding to thecalibrator from the top of the gradient

Slope_(gradient) is the slope (% per mm) of the gradient, or thedifference between the lowest and the highest gel concentrations (6% forthe top gradient and 22% for the bottom gradient) divided by the heightof the gel. The actual height of each gradient can be determined by theoperator for each gel using the scanner.

2-8% gradient: with Lp(a) C_(gel)=30.40×[2+(9.8)*(6/35)]=111.863

with LDL C_(gel)=25.70×[2+(13.7)*(6/35)]=111.748

8-30% gradient with HDL₂ C_(gel)=11.80×[8+(38.40-35.0)*(22/37)]=118.255

with HDL₃ C_(gel)=9.60×[8+(41.70-35.0)*(22/37)]=115.044

From this estimate of the gel constant, the diameter S_(unknown) of anyparticle can be calculated from the distance d_(known) from the top ofthe gel. It should be noted that the densitometer gives the position ofthe band as referenced to an arbitrary set point (typically, from theedge of the glass plate) and the distance to the top of the gradientmust be subtracted to obtain the true distance of migration into thegel. Furthermore, for the lower gradient gel, the actual height of theupper gel must also be subtracted:

S _(unknown) =C _(gel) /f(d_(unknown)).

With each gel, the calibrator plasma containing Lp(a), LDL, HDL₂ andHDL₃ of known particle diameters was always applied in 3 separate lanesincluding the two outside lanes and one toward the center of the gel.From the previously determined diameters of Lp(a) and LDL in thecalibrator plasma, 6 separate estimates (2 calibrators×3 lanes) wereobtained for the gel constant of the 2-8% gradient. The mean value wasused to determine the diameters of LDL and Lp(a) in the unknown samples.Similarly, the diameters of HDL₂ and HDL₃ in the calibrator plasmaallowed the calculation of 6 estimates for the gel constantcorresponding to the 8-30% gel gradient and the mean value was used incalculating the HDL particle diameter.

Lipoprotein Isolation

To examine the characteristics the different class of lipoproteins inthis system, individual fractions were isolated by ultracentrifugationfrom freshly collected plasma. VLDL was recovered in the supemate of the1.006 spin (24-hr spin at 39,000 RPM and 10° C.) using the SW 40Swinging bucket rotor (Beckman Instruments, Palo Alto, Calif.). LDL wasrecovered in the density range of 1.019-1.063 gm/ml by sequentialultracentrifugation. Lp(a) was isolated by density ultracentrifugationusing a modification of the method reported in the paper“Physico-chemical properties of apolipoprotein(a) and lipoprotein(a-)derived from the dissociation of human lipoprotein(a),” Fless et al., J.Biol. Chem. 261: 8712-8718 (1986). In brief, 1 ml of plasma was adjustedto a density of 1.050 gm/ml using solid KBr and layered with 12 ml of a1.040 density solution. Ultracentrifugation was performed at 35,000 RPMfor 15 hrs (10° C.) using the SW 40 swinging bucket rotor. The fractioncorresponding to Lp(a) was removed by careful aspiration and confirmedby ELISA.

In order to compare our estimates obtained for HDL-sized particles withthis new procedure, we used LpA-I and LpA-II/A-II fractions which hadbeen isolated by immunoaffinity chromatography as discussed in the paper“Altered particle size distribution of apoA-I-containing lipoproteins insubjects with coronary artery disease,” Cheung et al., J. Lipid. Res.32: 383-397 (1991) and generously donated by Dr. Marian Cheung of theNorthwest Lipid Research Laboratory. The diameters of the major proteinfractions in these HDL subclasses had been determined using the 4-30%gel available from Alamo Gels, Inc (San Antonio, Tex.). By using thesepurified HDL subfractions to compare the estimates of particle diametersobtained by the two gels we can be sure that only peaks corresponding toHDL proteins are visualized after staining with Coomassie.

Results

Reproducibility of the S-GGE 2.8/8.30 for Lipoprotein Particle Diameter

FIG. 8A illustrates the reproducibility of the linear gradient acrossthe 21 lanes of the gel as demonstrated by identical distances ofmigration across all lanes for all four major lipoprotein bands. FIG. 8Bpresents the actual particle diameters for obtained for LDL and Lp(a)when the calibrator plasma was applied in multiple lanes of 5 differentgels. Only 9.4% (3/32) of the individual particle diameter estimates forLDL were outside 1 SD of the mean and none were outside 2 SD. For Lp(a),12.5% (4 out of 32 lanes) of the particle diameter estimates from 5separate gels were outside 1 SD of the mean. None were outside 2 SD. Forthe HDL subfractions, 18.8% (6 out of 32 lanes) were outside 1 SD and6.2% (2 out of 32) were outside 2 SD (FIG. 2C). Table 1 presents themean values and fractional standard deviation (100×SD/Mean) for thedistance from the top and for the calculated particle diameter forLp(a), LDL, HDL₂ and HDL₃ from 16 lanes on the same gel (within run) aswell as values from 65 different gels (between runs).

The reproducibility of the gel constants (mean+SEM for the most recentseries of 100 gels) was obtained for both the 2-8% (C_(gel)=113.5+0.45)and the 8-30% linear gradients (C_(gel)=116.43±0.28). There was nostatistical difference between the two estimates for the C_(gel) fromthe lower and upper gradient gels as assessed by un-paired t-test. Theseempirical results further support the earlier observation that the poresize at any point in a linear gradient is defined solely by the gelconcentration, independent of the range of the gradient prepared.

In order to examine the effect of the pre-staining procedure on theelectrophoretic mobility of the lipoproteins, whole plasma aliquots froma single donor were incubated in the staining solution for 15 min, 30min, 1 hr and 2 hrs at room temperature and electrophoresed in adjacentlanes. As shown in FIG. 9A, there was no change in the position of anybands corresponding to the 4 major lipoprotein fractions. Table 2presents the actual particle diameters for 6 separate incubations at 15min, 1 hour and 2 hour for a freshly collected plasma sample whichdisplays all 4 major lipoprotein bands. There was no statisticaldifference in particle size with the different incubation times.

FIG. 9B illustrates the reproducibility of the position of LDL bands asdifferent concentrations of LDLc were applied. With the present protocolfor pre-staining and electrophoresis, particle size determination forLDL can be determined from a sample with an LDLc concentration of 40mg/dL. While there appeared to be a dose-dependence between LDLc and theintensity of the stained LDL bands, we were not able to establish areliable standard curve between the areas under the LDL peaks asdetermined from the densitometer and the concentrations of LDL whenplasma samples from different donors were analyzed. This is in contrastto the reported data obtained with protein staining. Differences in thelipid composition of LDL among individuals could account for thevariability in the stain intensity for different preparations of LDL.

In contrast to the sharp bands characteristic of LDL, small VLDL can beshown to have significantly broader peaks which are poorly stained (FIG.9C). For this experiment, VLDL from a normotriglyceridemic donor(CHOL=195, TG=80, HDLc=56 and Lp(a)<0.1 mg/dL) was isolated byultracentrifugation using the SW40 swinging bucket at density d<1.006gm/ml. In contrast to the band corresponding to 40 mg/dL of LDLc (Lane4, FIG. 9B), the band corresponding to 50 mg/dL of VLDL-cholesterol(Lane 3, FIG. 9C) was barely visible under identical stainingconditions. As shown, the band corresponding to VLDL from thisnormotriglyceridemic individual was slightly larger than Lp(a) in ourcalibrator (Lane S) and was significantly more heterogeneous asindicated by the broadness of stained band. In our hands, VLDL isolatedby ultracentrifugation of whole plasma obtained from individuals withfasting TG of 350 mg/dL or greater can be visualized as a dark stain atthe top of 2% gel suggesting that these particles were too large toenter the gel matrix. Using the value of gel constant calculated for the2-8% gel and Eq. 6, we would predict that only particles with diameterless than 55 nm would be able to enter the gradient.

Identification of Lp(a)

The identity of the Lp(a) band was confirmed by several approaches.First, the stain characteristics of the Lp(a) band are different fromthat of VLDL (FIG. 9C). The Lp(a) band is sharper and more comparable tothat of LDL than that of VLDL. Secondly, isolated fractions of Lp(a) canbe shown to correspond to distinct peaks upon electrophoresis in theS-GGE 2.8/8.30 gel with migration distance identical to thecorresponding peaks obtained with whole plasma (FIG. 10). And thirdly,analysis of postprandial plasma samples from an individual demonstratedthat the peak corresponding to Lp(a) was unchanged while the area underthe peak corresponding to remnants increased at 2 hours postprandiallybefore returning to fasting level after 10 hours (FIG. 11).

FIG. 10A illustrates actual gel scans for a plasma sample with a highconcentration of Lp(a). In FIG. 10B, we present the gel scan of apartially purified Lp(a) isolated by density gradientultracentrifugation using the SW40 swinging bucket as previouslydescribed by Fless et al. paper. The position of the Lp(a) peak was28.20 mm in whole plasma as compared to 28.52 mm for the isolated Lp(a)from the edge of the plate. This corresponds to a distance of 8.2-8.5 mmfrom the top of the gel gradient. The gel scan in FIG. 10C illustrates asingle peak for LDL isolated by ultracentrifugation in the density range1.020-1.063 g/ml from the same plasma. In all samples with Lp(a)concentrations of 35 mg/dL or greater as determined by ELISA, we haveconsistently been able to visualize a band at approximately 8.2-8.75 mmfrom the top of the gel corresponding to a particle diameter in therange of 27 to 30 nm.

To further demonstrate the ability of the gel to resolve small VLDL andremnants from Lp(a), we examined non-fasting plasma from an individualwith a distinct Lp(a) band present in fasting plasma (FIG. 11A). Asshown in FIG. 11A, a distinct peak corresponding to Lp(a) was noted thatwas slightly larger than LDL and a subpopulation of even largerparticles appeared as a shoulder (Peak 1) to the left of the Lp(a) peak.Peak 1 was not visible in the gel scans from the plasma of mostindividuals with normal TG levels (<100 mg/dL, FIG. 10). Postprandialplasma samples collected following the consumption of a standardizedliquid test meal containing fat and cholesterol (19,20) were subjectedto electrophoresis (FIGS. 11(B)-(D)). At 2 and 4 hours following thetest meal, when postprandial plasma TG were expected to increase, thisshoulder associated with larger lipoprotein particles clearly became adistinct peak (Peak 1) with increasing areas (FIGS. 11B and C). Theposition of this band, i.e. the diameter of this lipoprotein fraction,is not changed in postprandial plasma. By 10 hour after the test meal,as plasma TG returned to fasting level, this band of larger lipoproteinparticles was reduced back to a shoulder to the left of the Lp(a) peak(FIG. 11D). The particle diameter corresponding to this peak isestimated to be 36.7 nm. We postulate that this peak of largerlipoproteins corresponded to TG-rich lipoproteins and their remnantswhich were generated during postprandial lipemia. This shoulder isdemonstrable in only a subset of the individuals studied with the oralfat load and its presence does not appear to be associated with fastingTG levels. Additional experiments are ongoing to further characterizethe nature of this lipoprotein peak. It is clear, however, that thesharp band in the size range from 27 to 30 nm must correspond to Lp(a)and not to IDL or other TG-rich remnant lipoproteins which wouldtypically have larger diameters ranging from 33.5 to 39 nm.

HDL Particle Size: Comparison with SFBR 3/31 Gel

In order to compare the diameters of HDL particles obtained with our gelsystem and the conventional Pharmacia PAA 4/30 gel, we examined LpA-Iand LpA-I/A-II fractions isolated by immunoaffinity chromatography. Asmentioned above, these lipoproteins fractions were kindly provided byDr. Marian Cheung (Northwest Lipid Research Laboratory, University ofSeattle, Seattle, Wash.) and the corresponding diameters were determinedusing the 3-31% gels (SFBR 3/31, Alamo Gels, Inc., San Antonio, Tex.)following protein staining. FIG. 12A illustrates the lipid-stained bandsobtained for whole plasma, LpB, LpA-I and LpA-I/A-II for one individual.In contrast to the protein-stained gels disclosed, for examples, in thepaper “Altered particle size distribution of apoA-I-containinglipoproteins in subjects with coronary artery disease,” Cheung et al.,J. Lipid. Res. 32: 383-397 (1991) and the paper “High densitylipoproteins and coronary atherosclerosis: A strong inverse relationwith the largest particles is confined to normotriglyceridemicpatients,” Johansson et al., Arterioscl Thromb 11: 177-182 (1991), onlyone or two major bands could be visualized with the lipid stain. Formost samples, two lipid-rich bands can always be identified for LpA-Iand only one for LpA-I/A-II. Only the major bands with the greatest areaunder the peak based on the protein stain were selected in thiscomparison. The two estimates of HDL particle diameters for LpA-I andLpA-I/A-II from 11 individuals were highly correlated (r=0.978 for atotal of 37 peaks). We used this linear regression equation to calculatethe adjusted particle diameters of the HDL₂ and HDL₃ in our calibrator.A new gel constant was derived using these adjusted diameters and FIG.12B illustrated the correlation between the particle diameters for HDLsubfractions determined on the S-GGE 2.8/8.30 using the gel constantapproach and the values obtained by Dr. Cheung using the SFBR 3/31 geland the Rf approach. The slope of the linear regression was 0.948 withan intercept of 0.4 nm (r=0.97).

Effect of Sample Storage on LDL and Lp(a) Particle Diameter

FIG. 13 illustrates the reproducibility of lipoprotein particle sizebetween fresh samples and frozen samples from a group of 52 individualsincluding normolipidemic controls and subjects with varying degrees ofhyperlipidemia. Fresh plasma samples were analyzed as they wereavailable (within 7 hrs of collection) using as many as 20 separategels. Frozen samples were analyzed at the end of an 8-month storageperiod using four separate gels within a 2-week period. The mean (±SD)LDL particle diameter for the group was 25.7 nm (±0.807) based on theanalysis of freshly isolated plasma and 25.3 nm (±0.693) when analyzedfrom frozen plasma samples. There was no statistically significantdifference between the two measurements by two-tailed paired t-test. Themean (±SD) ratio of LDL diameter between the fresh and the frozenestimate was 1.0077 (±0.028). In 26.9% (17 of 51) of the samples, theratio of the two estimates of LDL particle diameters (fresh vs. frozen)was greater than 3.27% (outside 1 SD) following a single freeze-thawcycle. For 7.7% of the plasma samples (7 of 52), the ratio of the sizeestimates for LDL was greater than 6% (outside 2 SD) following a singlefreeze-thaw cycle. One third of the samples (16 of 52) were actuallystored for 12 months prior to re-analysis. Since there were nodifferences in the estimates for particle diameter with longer storage,results from these samples were included in the final analysis.

In the present report we have described a protocol for the preparationof a gradient gel system consisting of two linear gradients, an 8-30%gradient for particles in the range of HDL and a 2-8% gradient for LDLand larger lipoprotein particles. The present system offers severaladvantages over existing systems previously described in the literature.Using commonly available gel casting equipment and conventionalelectrophoretic supplies, this protocol allows the reproduciblepreparation of gradient gels which can accommodate up to 21 samples pergel, or 42 samples per run with a dual-gel electrophoretic chamber. Theprotocol allows the application of pre-stained plasma samples and thegel can be scanned for particle size immediately at the end of theelectrophoretic procedure. Elimination of the extensive staining andde-staining steps after electrophoresis should also minimize the need tohandle the gel preventing any artifact that these steps may introduce.More importantly, the use of a lipid stain allows the specificvisualization of only the lipoprotein fractions present in whole plasma.

Although the present invention has been described with reference tospecific details of certain embodiments thereof, it is not intended thatsuch details should be regarded as limitations upon the scope of theinvention except as and to the extent that they are included in theaccompanying claims. Many modifications and variations are possible inlight of the above disclosure.

For example, a computer can be used to coordinate the motion of themovable arm and the rate of flow according to the width of the gel toyield better gradient gels.

What is claimed is:
 1. A method for making a gel comprising the stepsof: a. holding a solution in a reservoir; b. transferring the solutionto a dispensing tip; c. delivering the solution to a gel chamber with alongitudinal axis by moving the dispensing tip along the longitudinalaxis in a continuous motion; and d. curing the delivered solution in thegel chamber to form the gel.
 2. A method for making a gel, comprisingthe steps of: a. holding a plurality of solutions in a plurality ofreservoirs; b. forming a fluid of at least one solution from theplurality of solutions; c. transferring the fluid to a dispensing tip;d. delivering the fluid to a gel chamber with a longitudinal axis bymoving the dispensing tip along the longitudinal axis in a continuousmotion; and e. curing the delivered fluid in the gel chamber to form thegel.
 3. The method of claim 2, wherein each solution of the plurality ofsolutions is a polyacrylamide solution with a unique concentration.
 4. Amethod for making a gradient gel, comprising the steps of: a. holding afirst solution at a first reservoir and a second solution at a secondreservoir; b. connecting the first reservoir and the second reservoirwith a channel; c. connecting an outlet with the second reservoir,wherein the outlet communicates with the channel so that a fluid of thefirst solution and the second solution is formed at the outlet; d.transferring the fluid to a dispensing tip; e. delivering the fluid to agel chamber with a longitudinal axis by moving the dispensing tip alongthe longitudinal axis in a continuous motion; and f. curing thedelivered fluid in the gel chamber to form the gradient gel.
 5. Themethod of claim 4, wherein the first solution is a polyacrylamidesolution with a first concentration.
 6. The method of claim 5, whereinthe second concentration is no less than the first concentration.
 7. Themethod of claim 4, wherein the second solution is a polyacrylamidesolution with a second concentration.
 8. The method of claim 7, whereinthe second concentration is no less than the first concentration.
 9. Asegmental linear gel made according to the method of claim
 4. 10. Amethod for making a plurality of gels, comprising the steps of: a.holding a plurality of solutions in a plurality of reservoirs; b.forming a plurality of fluids, each fluid having at least one solutionfrom the plurality of solutions; c. transferring the plurality of thefluids to a plurality of dispensing tips, each dispensing tip having onefluid therein; d. delivering the plurality of the fluids to a pluralityof gel chambers positioned in parallel thereby defining a longitudinalaxis by moving the dispensing tips along the longitudinal axis, so thateach of the plurality of the dispensing tips delivers one fluid thereinin motion into one of the gel chambers; and e. curing the deliveredfluids in the plurality of the gel chambers to form the plurality of thegels.
 11. The method of claim 10, wherein each solution of the pluralityof solutions is a polyacrylamide solution with a unique concentration.12. A segmental linear gel, comprising: a. a first segment comprising alinear polyacrylamide gradient of high to medium polyacrylamideconcentration; and b) a second segment comprising a linearpolyacrylamide gradient of medium to low polyacrylamide concentration,wherein the second segment is on top of and in continuous communicationwith the first segment.